1. Field of the Invention
This invention relates to a method for producing an anabolic or growth promoting state in a mammal. More specifically, this invention is directed to the use of a complex of IGF-I and one or more of its binding proteins to produce an anabolic state, including enhancing whole body and bone growth.
2. Description of Related Disclosures
In the circulation, in other body fluids, and in media conditioned by cultured cells, the somatomedins (IGF-I and IGF-II) are bound to specific high-affinity carrier proteins that have been implicated as modulators of IGF actions. The history of IGF binding proteins (BPs) dates back to 1984 when the existence of specific somatomedin carrier proteins in serum was first shown. Hintz, Clin. Endocrinol. Metab., 13: 31-42 (1984). Four distinct IGF BPs have now been cloned and sequenced, and in addition, several other, not yet thoroughly characterized, BP species have been identified in various tissues. See, e.g., Baxter and Martin, Prog. in Growth Factor Res., 1: 49-68 (1989); Roghani et al., FEBS Lett, 255: 253-258 (1989); Bautista et al., Clinical Res., 38: PA117 (1990). On the basis of the sequences it became evident that many of the previously recognized BPs, known by different names, were in fact the same, falling into a defined number of classes of cloned BPs. To clarify the present status of these BPs, the Workshop on IGF Binding Proteins, held in Vancouver, Canada, June 1989, proposed the names IGFBP-1, IGFBP-2, and IGFBP-3 for the binding proteins with defined sequences. Ballard et al., Acta Endocrinol. (Copenh), 121: 751-752 (1989). The consensus at the Workshop was that other, incompletely characterized IGFBPs be referred to by size and origin until sequenced. Since that time, another IGFBP, namely, IGFBP-4, has been sequenced, as described below.
Amniotic fluid was the first source from which IGFBP-1 was detected Chochinov et al., J. Clin. Endocrinol Metab., 44: 902-908 (1977). The protein has been purified also from tissue extract of fetal and maternal placenta and named placental protein. Kiostinen et al., Endocrinology, 118: 1375-1378 (1986). The mature protein contains 234 amino acids, predicting a molecular mass of 25.3 kD. Lee et al., Mol. Endocrinol., 2: 404-411 (1988); WO 89/09792 published 19 October 1989. IGFBP-1 migrates on SDS-PAGE at 28-35 kD depending on the stage of reduction. IGFBP-1 is a minor binding protein in serum and contains the unsaturated serum IGF-binding sites. Serum levels are inversely dependent on insulin and have a marked diurnal variation, the levels being highest early in the morning. These levels increase in pregnancy up to several hundred .mu.g/1, and amniotic fluid levels are up to 1000-fold higher than those in serum.
Carrier proteins of the IGFBP-2 class have been isolated from human fetal liver and rat and bovine cell lines. Binkert et al., EMBO J., 8: 2497-2502 (1989); Rosenfeld et al., J. Clin. Endocrinol. Metab., 70: 551-553 (1990). In humans, the mature form contains 289 amino acids and has an apparent molecular mass of 31-40 kD, depending on the stage of reduction on SDS-PAGE. In humans high IGFBP-2 levels have been found in the cerebrospinal fluid. The abundance of this protein in fetal tissue suggests that it has a role in regulating development. IGFBP-2 preferentially binds IGF-II.
The majority of serum IGFs are bound to a BP composed of two parts forming a complex of molecular mass 125-150 kD. IGFBP-3 is the IGF binding subunit (.beta.-subunit) in this complex. Baxter and Martin, Proc. Natl. Acad. Sci. USA, 86: 6898-6902 (1989). It is an acid-stable glycoprotein appearing on SDS-PAGE as a major and minor band, corresponding to 53 and 47 kD, respectively. The other components in the complex are the acid-labile, non-IGF-binding subunit (.alpha.-subunit) with a molecular mass of 84-86 kD [Baxter, WO 90/0569], and IGF-I or IGF-II (.gamma.-subunit). Sequencing of the cloned cDNA for IGFBP-3 (previously known as IGFBP-53) predicts a molecular mass of 28.7 kD for the non-glycosylated protein and reveals that IGFBP-3 shares 33% sequence identity with IGFBP-1. Wood et al., Mol. Endocrinol., 2: 1176-1185 (1988); WO 89/09268 published Oct. 5, 1989.
Most recently, a 25-kD IGFBP-4 has been isolated from cultured human osteoblast-like TE89 osteosarcoma cell conditioned media and sequenced. Mohan et al., Proc. Natl. Acad. Sci. USA, 86: 8338-8342 (1989). A similar, if not identical, IGFBP was isolated from human prostatic carcinoma cells and sequenced. Perkel et al., J. Clin. Endocrin. and Metab., 71: 533-535 (1990)]. Another similar IGFBP was identified in adult rat serum. Shimonaka et al., Biochem. Biophys. Res. Comm., 165: 189-195 (1989).
The levels of IGFBP in adult serum have been found to reflect the growth hormone (GH) status of individuals who are either GH-deficient or acromegalic. Thus, high levels of IGFBP-3 correlate with high levels of GH. Martin and Baxter, J. Clin. Endo. and Metabol., 61: 799-801 (1985). Under normal conditions about 95-98% of the IGF-I in human plasma is bound to the IGFBPs. Studies on size-fractionated human serum, subjected to IGF-I RIA after extraction of each fraction to remove binding activity, have indicated that 72% of the endogenous peptide is associated with the 150-kD fraction and 25% with the 50-kD fraction. Daughaday et al., J. Clin. Endocrinol. Metab., 55: 916-921 (1982).
The literature has ascribed to IGFBP-3 both a passive role as a carrier of IGF-I extending its circulatory half-life and an active role as a promoter of IGF-I activity. For example, it has been disclosed by BioGrowth, Inc. that IGFBP-3 significantly accelerates healing in an animal wound-healing model and that the complex of IGF-I and IGFBP-3 stimulates cortical and trabecular bone growth in rats in preliminary experiments, suggesting that the BP may be useful in treating osteoporosis. See Bioventure View, Vol. IV, No. 1 (Jan. 31, 1989), pages 19-20. See also EP 294,021 and 375,438 to BioGrowth, Inc. disclosing use of IGFBP-3 in conjunction with IGF-I or -II to treat diseases such as osteoporosis and human GH deficiency, and to heal wounds and increase animal growth, including delivery to bony tissues to stimulate bone growth (see, e.g., p. 8 of EP 294,021 and p. 11 of EP 375,438). See also WO 90/00569 published Jan. 2, 1990. No data are provided for these speculative uses. There is one suggestion by BioGrowth scientists that IGFBP-3 (called IGF-CP) apparently increases IGF-directed bone growth in rats. Talkington-Verser et al., Proceed. Intern. Symp. Control. Rel. Bioact. Mater., 16: 223-224 (1989). However, no protocols or data are provided.
It has also been disclosed that a 28-kD IGFBP from human placental and hepatoma cDNA libraries administered together with IGF-I, IGF-II, or other growth factors or formulated as common preparations for topical use in therapeutic devices useful for healing wounds or bones or for treating osteoporosis might be valuable for a steady, controlled release of the somatomedins in such devices. WO 89/08667, published Sep. 21, 1989, pages 8-9.
Another human BP equivalent to that of rat BRL-3A is reported in EP 369,943 published May 23, 1990 to be useful in combination with an IGF to treat, e.g., osteoporosis, Laron-type dwarfism, anemias, hypopituitarism, and wounds (see col. 15). IGF-I and -II BPs having IGF potentiating and inhibiting activities are described in WO 89/09792 published Oct. 19, 1989.
Further, based on studies using baby hamster kidney and human skin fibroblasts, it has been suggested that IGFBP acts as a reservoir, releasing continuously low amounts of IGF-I and thus creating a steady-state situation of receptor occupancy, which appears to be a better mitogenic stimulus than temporary large concentrations of IGF-I. Blum et al., Endocrinology, 125: 766-772 (1989).
However, several recent reviews cast further doubt on the precise biological activities of the IGFBPs. For example, Zapf et al., Growth Factors: From Genes to Clinical Application, Karolinska Nobel Conference Series, Eds. Vicki Sara et al., (Raven Press 1990), p. 227] states that inhibitory as well as enhancing effects of IGF carrier proteins on IGF actions have been observed in vitro, citing DeMellow et al., Biochem. Biophys. Res. Comm., 156: 199-204 (1988); Elgin et al., Proc. Natl. Acad. Sci. USA, 84: 3254-3258 (1987); Knauer and Smith, Proc. Natl. Acad. Sci. USA, 77: 7252-7256 (1980); Meuli et al., Diabetologia, 14: 255-259 (1978); Schweiwiller et al., Nature, 323: 169-171 (1986). Zapf et al. further state that it is still unknown whether the different IGFBP species known thus far differ with respect to inhibiting or enhancing the growth promoting effects of IGF.
On page 241 of the same book, Hall et al. state that, in general, IGFBP-1, similar to IGFBP-3, is found to inhibit IGF-I stimulation of amino acid uptake and DNA synthesis, citing, inter alia, Walton et al., P.S.E.B.M., 190: 315-319 (1989).
A 1988 review article reports that despite increasing interest in IGFBPs in recent years, their functions are still poorly understood. Baxter, Comp. Biochem. Physiol., 91B, 229-235 (1988), p. 232-233. Baxter points to some evidence that association with BPs may not always inhibit the activity of the IGFs and that cell types producing the BPs might be able to enhance their IGF responsiveness in an autocrine fashion. Examples cited are that some high molecular weight complexes from human plasma retain biological activity in rat adipocyte assays for insulin-like activity, cultured human fibroblasts secrete a BP of 35 kD that increases cell IGF binding, and a pure preparation of amniotic fluid BP significantly potentiates the effect of IGF-I in stimulating DNA synthesis in porcine smooth muscle cells and fibroblasts from various species. Furthermore, it has been shown that IGFBP-3 blocks the hypoglycemic action of IGF-I when administered subcutaneously together with the IGF-I in a 1:1 ratio. Spencer et al., 2nd International Symposium on Insulin-Like Growth Factors/Somatomedins, January 12-16, 1991, program and Abstracts p. 269.
Another view is that IGFBPs are produced locally in all tissues to concentrate locally produced IGF-I near cells requiring the IGF-I, reducing the active role of IGF-I bound to BPs and IGF-I circulating in the blood. Isaksson et al., Endocrine Reviews, 8: 426-438 (1987). It has been reported, for example, that IGF-I is produced locally in bone by GH [Nilsson et al., Science, 233: 571-574 (1986)], and GH receptors have been found on chondrocytes. Nilsson et al., J. Endocr., 122: 69-77 (1989).
Furthermore, the recent work of Conover, 72nd Annual Meeting of Endocrine Society, Prog. Abstract 186 (June 1990) shows in vitro that the activity of the IGFBPs, in enhancing the activity of IGF-I, is dependent on cells being exposed to the BPs alone. There was no response to IGF-I in cells incubated with pre-mixed BP and IGF-I. If the BP was incubated with the cells by itself, followed by addition of IGF-I, the activity of the added IGF-I was enhanced. These data suggest that co-mixing IGF and a IGFBP and co-injecting the complex would not result in an enhancement of the activity of the IGF-I.
It is an object of the present invention to provide a specific method for promoting the growth of mammals by administering through subcutaneous injection a complex of IGF-I and a IGFBP.
It is another object to provide a way to administer large doses of IGF-I to a patient without concern for hypoglycemia.
These and other objects will be apparent to one of ordinary skill in the art.